Project abstract Porphyromonas gingivalis (Pg), a gram-negative asaccharolytic anaerobe, is a major causative pathogen of chronic periodontitis. Pg not only causes tooth loss, but is also associated with increased risk for other systemic diseases such as atherosclerosis. Endothelial cells provide an interface for the systemic dissemination of Pg. Pg evades endothelial cell defenses and persist within endothelial cells, thereby allowing chronic infections like periodontal disease and systemic diseases to develop. Pg persistence can also leads to endothelial dysfunction resulting in impaired inflammatory signals and atherosclerosis. Hence, it is critical to understand the mechanisms of Pg infection of endothelial cells. Our central hypothesis is that Pg invades endothelial cells and evades host defenses by regulating its gene expression and metabolic requirements to survive within endothelial cells. To test this hypothesis, we propose to employ an innovative approach of integrating transcriptomics and metabolomics to better understand the pathogenic nature of Pg and its interaction with the endothelial cells. This comprehensive approach of combining dual RNA-seq and metabolomics data will identify significant gene- metabolite integrated networks that are unique to Pg infection of endothelial cells. Consequently, these studies will define those metabolic changes that occur during intracellular adaptation and survival of Pg and provide much needed fundamental insights into periodontal disease and systemic diseases associated with Pg. In order to achieve this goal, we propose the following specific aims. Aim 1: To determine and compare the RNA expression patterns of P. gingivalis (W83, W83?0717 & W83?0717::0717) and host cells (HMVEC and HCAEC) during the invasion and persistence states. Aim 2: To define the metabolomic changes that occur during the invasion and persistence states of P. gingivalis (W83, W83?0717 & W83?0717::0717) in HMVEC and HCAEC cells. These studies will be the first to characterize the mRNA and metabolite patterns of both Pg and host cells during pathogenesis. The expected outcome of this work is a comprehensive understanding of what molecular mechanism(s) specific to gene-metabolite networks Pg utilizes during invasion and persistence states. The successful completion of the proposed studies will lay the groundwork for developing effective treatment strategies to prevent PD and its associated systemic diseases.